Regulation of energy metabolism and obesity by modulating b cell activating factor (baff, blys) or baff signaling

ABSTRACT

Provided herein are methods and compositions for modulating energy metabolism and weight in mammals, in particular by modulating thermogenesis associated with brown fat, including thermogenesis by brown fat or brown fat cells, adaptive thermogenesis by brown fat or brown fat cells, thermogenic capacity of brown fat or brown fat cells, or a combination thereof. More specifically, methods and compositions provided herein for treating or preventing obesity, or methods and compositions for identifying compounds effective for treating or preventing obesity are taught in connection with ligands such as B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), their receptors, and molecules that modulate the interactions between the ligands and receptors.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Application No. 61/438,964, REGULATION OFENERGY METABOLISM AND OBESITY BY MODULATING B CELL ACTIVATING FACTOR(BAFF, BLYS) OR BAFF SIGNALING, filed on Feb. 2, 2011, which iscurrently co-pending herewith and which is incorporated by reference inits entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with U.S. Government support on behalf of theNational Institute of Allergy and Infectious Diseases (NIAID), Grant No.R01 AI075159. The U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention disclosed herein generally relates to methods andcompositions for modulating obesity in an animal, particularly a human.The invention disclosed herein generally further relates to methods andcompositions for modulating the concentration and/or activity of B-cellactivating factor (BAFF) or a proliferation-inducing ligand (APRIL), ortheir receptors or signaling partners. Methods and compositions formodulating factors upstream and downstream of BAFF and/or APRIL are alsodisclosed.

BACKGROUND

Obesity is a major risk factor for development of insulin resistance,dyslipidemia, hypertension (together: “metabolic syndrome”), type 2diabetes, atherosclerosis, non-alcoholic fatty liver disease and diversecancers. Obesity has become epidemic in Westernized cultures.

What is needed in the art are methods and compositions for identifyingkey factors in regulating obesity, particularly those associated withregulation of energy metabolism and weight gain/loss. More specifically,what is needed in the art are methods and compositions for treating orpreventing obesity, or methods and compositions for identifyingcompounds effective for treating or preventing obesity.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions for modulating energymetabolism and weight in mammals. More specifically, provided herein aremethods and compositions for treating and preventing obesity, andmethods and compositions for identifying compounds effective fortreating or preventing obesity. Even more specifically, provided hereinare methods and compositions for treating or preventing obesity, andmethods and compositions for identifying compounds effective fortreating or preventing obesity taught in connection with B-cellactivating factor (BAFF), its receptors, and closely related molecules.

In one aspect, the present invention provides a method of treating orpreventing diet-induced obesity and/or one or more sequelae thereof in asubject. In particular, such methods comprise: administering to asubject in need of treatment or at risk for developing obesity and/orone or more sequelae thereof, a therapeutically effective amount of apharmaceutical composition to enhance thermogenesis associated withbrown fat or brown fat cells. In some embodiments, the pharmaceuticalcomposition comprises, for example, a recombinant BAFF or a variantthereof, a BAFF-based peptide or peptide analogue, a BAFF transcriptionregulator, a nucleic acid encoding at least a partial sequence of BAFF,a first factor upstream or downstream from the interaction between BAFFand a first receptor, a proliferation-inducing ligand (APRIL) that isrecombinant or a variant thereof, an APRIL-based peptide or peptideanalogue, an APRIL transcription regulator, a nucleic acid encoding atleast partial sequence of APRIL, a second factor upstream or downstreamfrom the interaction between APRIL and a second receptor, a regulator ofa BAFF-first receptor interaction or an APRIL-second receptorinteraction, or a combination thereof.

In some embodiments, thermogenesis associated with brown fat comprisesthermogenesis by brown fat or brown fat cells, adaptive thermogenesis bybrown fat or brown fat cells, thermogenic capacity of brown fat or brownfat cells, or a combination thereof.

In some embodiments, thermogenesis associated with brown fat or brownfat cells is enhanced by increasing the level or activity of BAFF.

In some embodiments, thermogenesis associated with brown fat or brownfat cells is further enhanced by increasing the level or activity ofAPRIL.

In some embodiments, the first receptor of BAFF is BAFF-receptor(BAFF-R), cytophilin ligand interactor (TACI) or a B-cell maturationantigen (BCMA).

In some embodiments, the pharmaceutical composition is a regulator ofBAFF-first receptor interaction, including but not limited to aBAFF-based peptide or an analogue thereof, a BAFF receptor analogue, aco-activator of BAFF-first receptor interaction, or a combinationthereof.

In some embodiments, the pharmaceutical composition is a recombinantBAFF or a variant thereof.

In some embodiments, the first factor upstream or downstream from theinteraction between BAFF, and the first receptor is radioprotectiveprotein 105 kDa (RP105).

In some embodiments, the pharmaceutical composition is administered to asubject via parenteral, topical, oral, or local administration,injection, or a combination thereof.

In some embodiments, the subject is a human in need of treatment or atrisk for developing obesity and/or one or more sequelae thereof.

In some embodiments, the method further comprises: identifying thesubject (e.g. a human) in need of treatment or at risk for developingobesity and/or one or more sequelae thereof.

In some embodiments, the BAFF transcription can be regulated by atoll-like receptor 4 (TLR4) agonist, a lipopolysaccharide (LPS),reactive oxygen species (ROS) production, NF-κB activation, p65,co-activator p300, or a combination thereof.

In some embodiments, the second receptor of APRIL is TACI or BCMA.

In some embodiments, thermogenesis associated with brown fat or brownfat cells is enhanced by increasing the level or activity of APRIL.

In some embodiments, thermogenesis associated with brown fat or brownfat cells is enhanced by increasing the level or activity of APRIL andBAFF.

In some embodiments, the pharmaceutical composition is a regulator of anAPRIL-second receptor interaction selected from the group consisting ofan APRIL-based peptide or an analogue thereof, an APRIL receptoranalogue, and a co-activator of an APRIL-second receptor interaction.

In some embodiments, the first receptor and the second receptor are thesame.

In some embodiments, the pharmaceutical composition is a recombinantAPRIL, a fragment of APRIL, an APRIL variant, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIGS. 1A-B illustrates that RP105-deficient mice are protected fromobesity and glucose dysmetabolism. Six to eight week-old male C57BL/6RP105−/− mice and wild type (WT) controls were placed on a high fat diet(HFD) or a regular chow diet (RD). FIG. 1A depicts mouse body weight. WTRD results are represented by white squares; WT HFD results arerepresented by white circles; RP105−/− RD results are represented byblack squares; RP105−/− HFD results are represented by black circles.FIG. 1B depicts fasting glucose levels, quantified after 10 weeks on thediet. After an 8 hour fast, blood glucose levels were quantified byglucometer. RD results are represented by white bars; HFD results arerepresented by black bars. Data represent means±SE in a singleexperiment, representative of 10 individual experiments; n=10-14mice/group. For FIG. 1A, AUC ANOVA P<0.01, Tukey's correction***P<0.001; for FIG. 1B, ANOVA P<0.0001, Tukey's correction *P<0.01.

FIGS. 2A-B illustrates the comparison between serum BAFF levels inRP105−/−, μMT and BAFF-Tg mice. FIG. 2A depicts a kinetic analysis ofserum BAFF levels in WT and RP105−/− mice. Mice were mock-challenged orchallenged with TLR4-specific E. coli lipopolysaccharide (LPS) (40 μg,i.p.), and serum BAFF levels were quantified kinetically. WT results arerepresented by white bars; RP105−/− results are represented by blackbars. Data represent means±SE in a single experiment; n=6 mice/group;unpaired, t test *P<0.05, **P<0.005. FIG. 2B depicts baseline serum BAFFlevels in RP105−/−, μMT, and BAFF-Tg mice. Data represent means±SE in asingle experiment; n=3-4 mice/group; ANOVA P<0.0001, Tukey's correction***P<0.0001, on log transformed data.

FIGS. 3A-D illustrates that BAFF overexpression is associated withprotection from weight gain and glucose dysmetabolism in diversetransgenic mice. Six to eight week-old male μMT, BAFF-transgenic, and WTmice (all on a C57BL/6 background) were placed on a HFD or RD. FIG. 3Adepicts mouse body weight. WT RD results are represented by whitesquares; WT HFD results are represented by white circles; μMT RD resultsare represented by black squares; μMT HFD results are represented byblack circles. FIG. 3B depicts fasting glucose levels, quantified after9 weeks on the diet. RD results are represented by white bars; HFDresults are represented by black bars. FIG. 3C depicts mouse bodyweight. WT RD results are represented by white squares; WT HFD resultsare represented by white circles; BAFF-transgenic RD results arerepresented by black squares; BAFF-transgenic FHD results arerepresented by black circles. FIG. 3D depicts fasting glucose levels,quantified after 12 weeks on the diet. RD results are represented bywhite bars; HFD results are represented by black bars. Data representmeans±SE. FIGS. 3A-B data are taken from a single experiment,representative of 4 individual experiments; n=5-6 mice/group. FIGS. 3C-Ddata are taken from a single experiment; n=3-4 mice/group. For FIGS. 3Aand 3C, AUC ANOVA P<0.01, Tukey's correction *P<0.05, ***P<0.001; forFIGS. 3B and 3D, ANOVA P<0.0001, Tukey's correction *P<0.01.

FIG. 4 illustrates the comparison of the response to HFD stress acrossgenotypes. The figure depicts the difference in mean percent weight gain(compared with WT controls) after 12 weeks on the diet in mice; datawere taken from the experiments depicted in FIGS. 1 and 3. RD resultsare represented by white bars; HFD results are represented by blackbars.

FIG. 5 illustrates that BAFF stimulates brown adipocyte respiration invitro. An immortalized mouse brown adipocyte cell line wasdifferentiated and subsequently stimulated with rBAFF (2 μg/ml) ormock-stimulated, and the oxygen consumption rate was quantified using anXF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica,Mass.) at the times indicated after BAFF addition; all conditions weremeasured at the same time. Data represent means±SE in a singleexperiment; ANOVA P<0.005, Tukey's correction *P<0.05, **P<0.01.

FIG. 6 illustrates that brown fat mitochondria from RP105-deficient miceexhibit increased uncoupled respiration. WT and RP105−/− C57BL/6 malemice were fed a HFD for 24 weeks. Uncoupled respiration (pyruvate/malateinduced, GDP inhibitable respiratory activity) was quantified inmitochondria isolated from brown adipose tissue using an XF24extracellular flux analyzer (Seahorse Bioscience). WT results arerepresented by white bars; RP105−/− results are represented by blackbars. Data represent means±SE, normalized to Vmax (oxygen consumptiondriven by FCCP); n=4 mice/group; *P=0.002 (Student's t test).

FIGS. 7A-E depict exemplary amino acid sequences of BAFF (SEQ ID NO 1-3)and exemplary amino acid sequences of APRIL (SEQ ID NO. 4 and N0.5).

FIG. 8 illustrates an exemplary embodiment, demonstrating that adaptivethermogenesis was compared in RP105−/− and WT mice on a HFD. RP105−/−and WT mice, both on a C57BL/6 background, were placed on a HFD (60%kcal from fat, 20% kcal from carbohydrate, 20% kcal from protein,Research Diets #D12492i) for 14 days. Mice were anaesthetized byintraperitoneal injection of ketamine/xylazine, and the basal rate ofoxygen consumption (at 33° C.) was quantified kinetically, at 3 minuteintervals for 24 minutes, by indirect calorimetry. Oxygen consumptioncontinued to be quantified kinetically after subsequent subcutaneouschallenge, depicted on the figure with an arrow, with 1 mg/kgnorepinephrine bitartrate. WT results are represented by open symbols;RP105−/− results are represented by closed symbols. Data representmeans+/−SE of an n=4-5 mice/experimental group; t test; *P<0.05.

FIG. 9 illustrates an exemplary embodiment, demonstrating that WTC57BL/6 mice were placed on either a HFD (60% kcal from fat, 20% kcalfrom carbohydrate, 20% kcal from protein, Research Diets #D12492i) or anRD (chow; 13.5% kcal from fat, 59% kcal from carbohydrate, 27.5% kcalfrom protein, Lab Diet #5010) for 14 days. Mice were subsequently given1 mg/kg recombinant BAFF or vehicle control (saline) by intraperitonealinjection 18 hours and 2 hours prior to quantification of oxygenconsumption. Mice were then anaesthetized by intraperitoneal injectionof ketamine/xylazine, and the basal rate of oxygen consumption (at 330°C.) was quantified kinetically, at 5 minute intervals for 25 minutes, byindirect calorimetry. Oxygen consumption continued to be quantifiedkinetically after subsequent subcutaneous challenge, depicted on thefigure with an arrow, with 1 mg/kg norepinephrine bitartrate. RD+salineresults are represented by open squares; RD+BAFF results are representedby closed squares; HFD+saline results are represented by open circles;HFD+BAFF results are represented by closed circles. Data representsingle mice/experimental group.

FIG. 10 illustrates that RP105-deficient mice maintain body coretemperature better during cold exposure. Six to eight week-old maleC57BL/6 RP105^(−/−) mice and WT controls were placed on a HFD for 16weeks. Animals were subsequently placed at 4° C., and body coretemperature was quantified. The mice were then subjected to 0-6 hours ofcold exposure, under lighted conditions; 6-12 hours cold exposure wasconducted under dark conditions. WT HFD results are represented by whitesquares; RP105^(−/−) HFD results are represented by black squares. Datarepresent means±SE in a single experiment, representative of 2individual experiments; n=5-6 mice/group; *P<0.05 (Student's t test).

FIG. 11 illustrates that RP105-deficient mice have increased uncouplingprotein 1 (UCP-1) expression in brown adipose tissue. Six to eightweek-old male C57BL/6 RP105^(−/−) mice and wild type (WT) controls wereplaced on a regular diet (RD) or a high fat diet (HFD) for 20 weeks, andUCP-1 expression was quantified by qRT-PCR. WT results are representedby white bars; RP105^(−/−) results are represented by black bars. Datarepresent means±SE in a single experiment, representative of 2individual experiments; n=6 mice/group; *P<0.05 (Student's t test).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.

As used herein, the term “pharmaceutical composition” encompasses anyprotein, peptide, peptide analogue, nucleic acid, or small molecule thatcan interfere with thermogenesis by brown fat. In particular, thosecompositions that can interfere with signaling pathways mediated byB-cell activating factor (BAFF) and/or a proliferation-inducing ligand(APRIL).

Obesity and RP105

Radioprotective protein 105 kDa (RP105) is an endogenous modulator ofToll-like receptor (TLR) signaling pathways. Originally described as a Bcell-specific molecule that drives proliferation when cross-linked byantibodies, it has been shown that: (a) RP105 expression mirrors that ofTLRs on myeloid and other cells; and (b) RP105 inhibits TLR4 signalingin primary myeloid cells and cell lines and regulates in vivo responsesto lipopolysaccharide (LPS). The data indicate that RP105 also regulatesenergy metabolism in response to caloric excess. As shown in FIG. 1A,RP105-deficient mice gain significantly less weight than wild type (WT)controls when subjected to stress induced by a high fat diet (HFD).RP105−/− mice eat the same amount as WT controls on a HFD and haveidentical food absorption (as quantified by bomb calorimetry). Thesedata indicate that RP105 regulates metabolic efficiency in response tocaloric excess, hence indicating that RP105 regulates energy metabolism.Adaptive thermogenesis by brown fat, which occurs in response to caloricexcess as well as cold stress, is dependent on the expression ofuncoupling protein 1 (UCP1). The data also indicate that: (a) brown fatfrom RP105−/− mice on a HFD express significantly more UCP1 mRNA than WTcontrols (data not shown); and (b) brown fat mitochondria from RP105−/−mice on a HFD exhibit significantly higher degree of uncoupledrespiration than brown fat mitochondria from wild type controls on aHFD.

Additional information on RP105 can be found, for example, in Divanovicet al., 2005, “Inhibition of TLR-4/MD-2 signaling by RP105/MD-1,”Journal of Endotoxin Research 11(6):363-368; Divanovic et al., 2005,“Negative regulation of Toll-like receptor 4 signaling by the Toll-likereceptor homolog RP105,” Nature Immunology 6(6):571-578, each of whichis hereby incorporated by references herein in its entirety.

Thermo Genesis by Brown Fat

In one aspect, BAFF modulates energy metabolism and metabolic efficiencyby modulating thermogenesis by brown adipose tissue (BAT, or brown fat).More specifically, regulation of thermogenesis by brown fat includes butis not limited to thermogenesis, adaptive thermogenesis and thermogeniccapacity by brown fat. Thermogenesis is the process of heat productionin organisms. While thermogenesis occurs in all cells and organisms;brown fat-associated thermogenesis occurs in mammals.

In some embodiments, thermogenesis encompasses those processes initiatedthrough locomotion and intentional movement of the muscles, includingbut not limited to exercise-associated thermogenesis (EAT), non-exerciseactivity thermogenesis (NEAT), and diet-induced thermogenesis (DIT).

In some embodiments, thermogenesis encompasses various types of adaptivethermogenesis caused, for example, by changes in the surroundingtemperature of the animal or changes in dietary intake.

In an animal, heat can be generated by the conversion of the chemicalenergy of ATP into kinetic energy, i.e. by shivering. Non-shiveringthermogenesis occurs in the brown fat that is present in most if not allmammals, including humans. In this process, substances such as freefatty acids (derived from triacylglycerols) inhibit thermogenin(uncoupling protein-1 or UCP1) by removing purine (in the form of ADP,GDP and others), which causes an influx of H⁺ into the matrix of themitochondria and bypasses the ATP synthase channel. This uncouplesoxidative phosphorylation, and the energy from the proton motive forceis dissipated as heat rather than producing ATP from ADP, which wouldstore chemical energy for the body's use. Thermogenesis can also beproduced by leakage of the sodium-potassium pump and the Ca²⁺ pump.Thermogenesis is contributed to by futile cycles, such as thesimultaneous occurrence of lipogenesis and lipolysis or glycolysis andgluconeogenesis.

Regulation of non-shivering, adaptive thermogenesis by brown fat isregulated by the adrenergic stimulation by the sympathetic nervoussystem, as well as by the central nervous system activities of thyroxineand leptin; the latter hormones stimulate thermogenesis by activatingthe sympathetic nervous system. See, for example, Cannon and Nedergaard,2010, “Thyroid hormones: igniting brown fat via the brain,” NatureMedicine 16:965-967, which is hereby incorporated by reference herein inits entirety. In some embodiments, the level of any or a combination ofthese indicators are used to monitor thermogenesis. For example,upregulation of the thermogenic response of mice to norepinephrinechallenge is a standard experimental test for the evaluation of adaptivethermogenesis. Alternatively, cold exposure can be used to induce andmeasure adaptive thermogenic capacity. See, for example, Skilling etal., 2010, “Brown adipose tissue function in short-chain acyl-CoAdehydrogenase deficient mice,” Biochem Biophys. Res. Commun.400(3):318-322; Lammoglia et al., 2010, “Effects of prepartumsupplementary fat and muscle hypertrophy genotype on cold tolerance innewborn calves,” Journal of Animal Science 77(8):2227-2233, each ofwhich is hereby incorporated by reference herein in its entirety.

An overview of the role of brown fat in adaptive thermogenesis can befound, for example, in Stock and Rothwell, 1983, “Role of brown adiposetissue thermogenesis in overfeeding: a review,” J. R. Soc. Med.76(1):71-73, which is hereby incorporated by reference herein in itsentirety. Additional information can be found, for example, inNedergaard and Cannon, 2010, “The Changed Metabolic World with HumanBrown Adipose Tissue: Therapeutic Visions,” Cell Metabolism11(4):268-272; Cannon and Nedergaard, 2011, “Nonshivering thermogenesisand its adequate measurement in metabolic studies,” The Journal ofExperimental Biology 214: 242-253, each of which is hereby incorporatedby reference herein in its entirety.

In accordance with the present invention, any compositions and/or means,which alter thermogenesis, e.g. thermogensis by brown fat, in an animalcan affect the weight status of the animal. The animal can be a human, acow, a horse, a sheep, a dog, a mouse, etc. In particular, anycompositions and/or means which can upregulate thermogenesis by brownfat (or brown fat cells), adaptive thermogenesis by brown fat (or brownfat cells), and/or the thermogenic capacity of brown fat cells (or brownfat cells), can be used to prevent or treat obesity. Similarly, anycompositions and/or means, which can downregulate thermogenesis by brownfat (or brown fat cells), adaptive thermogenesis by brown fat (or brownfat cells), and/or the thermogenic capacity of brown fat cells (or brownfat cells), can be used to prevent or treat excessive weight loss (suchas anorexia).

In some embodiments, modulation of thermogenesis is achieved bymodifying the function of brown adipocytes or brown adipose tissue.

Brown fat is one of two types of fat or adipose tissue found in mammals(the other being white adipose tissue). Brown fat is present in most ifnot all mammals, including humans, and is especially abundant innewborns and in hibernating mammals. BAFF and various homologues can befound in various mammals as well. Given the prevalence of BAFF and brownfat in different mammals and their important role in regulatingthermogenesis, compositions and/or methods targeting BAFF-mediatedthermogenesis by brown fat can serve as therapeutic options for diseasessuch as obesity and sequelae thereof.

Adaptive thermogenesis is defined as the regulated production of heatcaused by uncoupled respiration in brown fat, resulting in metabolicinefficiency and is believed to be a mechanism for modulating coldstress and dietary excess. In contrast to white adipocytes (white fatcells), which contain a single lipid droplet, brown adipocytes containnumerous smaller droplets and a much higher number of mitochondria,which contain iron and account for the brown color. Brown fat alsocontains more capillaries than white fat, since it has a greater needfor oxygen than most tissues.

BAT is a unique form of fat that is found in varying amounts throughoutthe mammalian genus. Bears, rodents, and other hibernating animals thatlive in cold weather have an abundance of this specialized fat. Humanshave small deposits throughout the body, although the content is muchhigher during infancy. BAT cells are smaller than normal storage cells,contain less fat, and are laden with mitochondria. These mitochondriahold a specialized uncoupling protein, namely uncoupling protein 1(UCP1, also known as thermogenin), that allows for uncoupling ofoxidative phosphorylation, leading to the production of heat but no ATP.

The adaptive thermogenic capacity of brown adipose tissue is inducedthrough two primary mechanisms, cold environment and excess foodconsumption. These stimuli drive the differentiation, proliferation, andfunction of brown fat adipocytes.

Thermogenesis by brown fat is, with basal metabolic rate andactivity-induced thermogenesis, one of the three components of dailyenergy expenditure. Thermogenesis by brown fat is thought to play a rolein the prevention or control of obesity. Additional details can befound, for example, in Nedergaard and Cannon, 2010, “The changedmetabolic world with human brown adipose tissue: therapeutic visions,”Cell Metab. 11:268-272; Enerbäck S. 2010, “Human brown adipose tissue,”Cell Metab. 11:248-252; and Tseng et al., 2010, “Cellular bioenergeticsas a target for obesity therapy,” Nature Reviews Drug Discovery9:465-482, each of which is hereby incorporated by reference herein inits entirety.

The mitochondria in a eukaryotic cell utilize fuels to produce energy(in the form of ATP). This process involves storing energy as a protongradient, also known as the proton motive force (PMF), across themitochondrial inner membrane. This energy is used to synthesize ATP whenthe protons flow across the membrane (down their concentration gradient)through the ATP synthase enzyme; this is known as chemiosmosis. Inwarm-blooded animals, body heat is maintained in part by signaling themitochondria to allow protons to run back along the gradient withoutproducing ATP. This can occur since an alternative return route for theprotons exists through UCP1 in the inner membrane, which facilitates thereturn of the protons after they have been actively pumped out of themitochondria by the electron transport chain. This alternative route forprotons uncouples oxidative phosphorylation, and the energy in the PMFis instead released as heat.

To some degree, all cells give off heat. However, brown adipose tissueis highly specialized for thermogenesis through uncoupled respiration.This is because each cell has a higher number of mitochondria comparedto more typical cells. In addition, these mitochondria contain UCP-1.

More details about the connection between brown fat and thermogenesis inhumans can be found, for example, in Cypess et al., 2009,“Identification and importance of brown adipose tissue in adult humans,”New Eng. J. Med. 360:1509-1517; Kuroshima 1993, “Brown adipose tissuethermogenesis as a physiological strategy for adaptation,” Japan J.Physiol. 43:117; van Marken Lichtenbelt et al., 2009, “Cold-activatedbrown adipose tissue in healthy men,” New Eng. J. Med. 360:1500-1507;Virtanen et al., 2009, “Functional brown adipose tissue in healthyadults,” New Eng. J. Med. 360:1518-1525, each of which is herebyincorporated by reference herein in its entirety.

In some embodiments, recruitment of adaptive thermogenic capacity andadaptive thermogenesis by brown fat occurs in response to changes indietary intake, including diet with caloric excess and diets that arecalorie rich but are limiting in essential nutrients.

Diet-induced-thermogenesis (DIT) has two essential components whichcontribute to energy expenditure. Immediately after eating and for aperiod of several hours thereafter, the body expends more energy tosupport the ingestion, digestion, and absorption of nutrients. Inaddition, the body produces heat in brown adipose tissue (BAT) due toincreased sympathetic nervous system activity. The latter is adaptivethermogenesis mediated by brown fat or brown fat cells.

Thus, in some embodiments, treatment of obesity can be a combinatorialapproach. That is to say, additional compositions and methods can beused in addition to those used for modulating BAFF-mediatedthermogenesis by brown fat.

In some embodiments, compositions and methods disclosed herein can beapplied to the context of regulating diet-related thermogenesis by brownfat.

In some embodiments, altering the expression of specific moleculesassociated with thermogenesis by brown fat can modify energy metabolismand/or control weight gain.

Additional information on energy metabolism (e.g. thermogenesis) and itsassociation with brown fat can be found, for example, in Tseng, Y. H.,et al., 2008, “New role of bone morphogenetic protein 7 in brownadipogenesis and energy expenditure,” Nature 454:1000-1004; Pfluger, P.T., et al., 2008, “Sirtl protects against high-fat diet-inducedmetabolic damage,” Proc. Natl. Acad. Sci. U.S.A. 105:9793-9798; Hofmann,S. M., et al., 2007, “Adipocyte LDL receptor-related protein-1expression modulates postprandial lipid transport and glucosehomeostasis in mice,” J. Clin. Invest. 117:3271-3282; Kirchner, H., etal., 2010, “Ghrelin and PYY in the regulation of energy balance andmetabolism: lessons from mouse mutants,” Am. J. Physiol. Endocrinol.Metab. 298:E909-919; Pfluger, P. T., et al., 2008, “Simultaneousdeletion of ghrelin and its receptor increases motor activity and energyexpenditure,” Am. J. Physiol. Gastrointest. Liver Physiol. 294:G610-618;Kirchner, H., et al., 2009, “GOAT links dietary lipids with theendocrine control of energy balance,” Nat. Med. 15:741-745; Collins, S.,et al., 2010, “Positive and negative control of UCP1 gene transcriptionand the role of beta-adrenergic signaling networks,” Int. J. Obes.(Lond.) 34 Suppl 1:S28-33; Shabalina, I. G., et al., 2010, “Uncouplingprotein-1 is not leaky,” Biochim. Biophys. Acta 1797:773-784, each ofwhich is hereby incorporated by reference herein in its entirety.

B-Cell Activating Factor (BAFF)

Provided herein are compositions and/or methods for enhancingthermogenesis by modulating cell-signaling pathways relating tothermogenesis by brown fat, for example, the BAFF signaling pathway andthe like. BAFF, also known as tumor necrosis factor (TNF) ligandsuperfamily member 13B is a protein that in humans is encoded by theTNFLSF13B gene. BAFF is also known as B lymphocyte stimulator (BLyS) andTNF- and APOL-related leukocyte expressed ligand (TALL-1), THANK, zTNF4,and the dendritic cell-derived TNF-like molecule (CD257 antigen; clusterof differentiation 257). BAFF is a type II transmembrane protein thatcan be proteolytically cleaved between Arg 133 and Ala 134 and releasedas a soluble protein.

BAFF is a cytokine that belongs to the TNF ligand family and is a ligandfor receptors TNFRSF13B/TACI (cytophilin ligand interactor),TNFRSF17/BCMA (B-cell maturation antigen), and TNFRSF13C/BAFF-R(BAFF-receptor). This cytokine acts as a potent B-cell activator. It hasbeen also shown to play an important role in the proliferation anddifferentiation of B-cells.

Human BAFF is a 285 amino acid (AA) peptide consisting of a 218 AA acidextracellular domain, a 21 AA transmembrane region, and a 46 AAcytoplasmic tail. BAFF is a glycoprotein, which undergoes glycosylationat residue 124. It is expressed as a transmembrane protein on variouscell types, including monocytes, dendritic cells, B-cells, adipocytes,and bone marrow stromal cells. The transmembrane form can be cleavedfrom the membrane to generate a soluble protein fragment. BAFF is thenatural ligand of three TNF receptors, namely BAFF-R, TACI, and BCMA,all of which are type III transmembrane proteins and have differingbinding affinities for BAFF. These receptors are expressed mainly onmature B lymphocytes. TACI is also found on a subset of T-cells and BCMAon plasma cells.

Receptors of BAFF and other related ligands in parallel signalingpathways can be found in a variety of cell/tissue type, includingadipocytes. Additional examples of receptors can be found, for example,in Alexaki et al., 2009, “Adipocytes as Immune Cells: DifferentialExpression of TWEAK, BAFF, and APRIL and Their Receptors (Fn14, BAFF-R,TACI, and BCMA) at Different Stages of Normal and Pathological AdiposeTissue Development,” The Journal of Immunology 183(9):5948-5956; Kim etal., 2009, “B cell activation factor (BAFF) is a novel adipokine thatlinks obesity and inflammation,” Exp. Mol. Med. 41(3):208-216, each ofwhich is hereby incorporated by reference herein in its entirety.

TACI also binds to APRIL, which is a protein similar to BAFF. BCMAdisplays an intermediate binding phenotype and will work with eitherBAFF or APRIL to varying degrees. Signaling through BAFF-R and BCMAstimulates B lymphocytes to undergo proliferation and to counterapoptosis. Signaling is also achieved through TACI. BAFF is expressed bymyeloid cells and by unidentified radiation-resistant cells, possiblystromal cells of secondary lymphoid organs. BAFF is either expressed atthe cell surface or released into a soluble form through cleavage by anuncharacterized furin. Members of the TNF family usually assemble astrimers, but soluble BAFF was crystallized both as a trimer (e.g. ahomotrimer) and as a virus-like structure resulting from the orderedassembly of 20 trimers through an unusually long loop of BAFF betweenbeta-sheets D and E (DE loop). Recent work has shown that a BAFF 60-meris also produced by cells expressing BAFF endogenously and that the60-mer was moderately more potent than the 3-mer at costimulatingBCR-induced thymidine uptake in primary B-cells. APRIL, a close homologof BAFF, can also co-stimulate B-cells but requires oligomerization todo so. For example, APRIL functions as a homotrimer,

The state of oligomerization can be another level of regulation. Forexample, it has been shown that, in contrast to BAFF-R, TACI wasunresponsive to the BAFF 3-mer yet provided the survival anddifferentiation signals when triggered by oligomeric forms of BAFF orAPRIL. BAFF 60-mer is one form of oligomeric BAFF that can activate TACIand that exists naturally in vivo.

A strong pH dependence of oligomerization state has been observed: at pH6.0 BAFF was 100% trimeric and at pH 7.4 100% 60-mer. Thus, it possibleto manipulate pH value anywhere between 6.0 and 7.4 in order to achievethe desired levels of trimer and 60-mer. In some embodiments,recombinant BAFF is genetically modified in order to preferentially formthe trimer or 60-mer. Methods for making BAFF trimers and BAFF 60-merscan be found, for example, in United States Patent Publication No.20090215071 to Cachero et al.; U.S. Pat. No. 7,553,930, each of which ishereby incorporated by reference herein in its entirety.

The role of BAFF in energy metabolism and control of weight can be foundin Examples 3-6. In particular, Example 3 (FIGS. 1-4) illustrates thatBAFF overexpression is associated with protection from weight gain andglucose dysmetabolism. The correlation between BAFF overexpression andweight gain can be observed across mice of different genotypes (Example4). Modifications of BAFF, either its level of expression or activity,are provided as methods for treating disorders in energy metabolism(e.g. obesity).

The present invention provides that increased BAFF in vivo is associatedwith protection from obesity. In particular, the protection isassociated with increased UCP-1 expression and increased uncoupledrespiration in brown fat mitochondria. It has been demonstrated in vitrothat BAFF directly increases brown adipocyte respiration. Morespecifically, the presence of BAFF stimulates brown adipocyterespiration in vitro. Example 5 shows that level of BAFF correlates withlevel of oxygen consumption rate. Example 6 further illustrates that theenhanced respiration level of brown fat cells (e.g. resulting fromstress induced by a HFD) corresponds with increased uncoupledrespiration in brown fat mitochondria. Example 9 further demonstratesthat BAFF treatment up-regulates norepinephrine-induced thermogenesis invivo. Thus, BAFF drives increased thermogenesis, thermogenic capacity,and/or adapative thermogenesis by brown fat.

Additional information on BAFF, its homologues and receptors, and theirfunctions can be found, for example, in Schneider, P. & Tschopp, J.,2003, “BAFF and the regulation of B cell survival,” Immunol. Lett.88:57-62; Groom, J. R., et al., 2007, “BAFF and MyD88 signals promote alupuslike disease independent of T cells,” J. Exp. Med. 204:1959-1971;Kalled, S. L., 2005, “The role of BAFF in immune function andimplications for autoimmunity,” Immunol. Rev. 204:43-54; Mackay, F. &Schneider, P., 2009, “Cracking the BAFF code,” Nat. Rev. Immunol.9:491-502; Kim, Y. H., 2009, “B cell activation factor (BAFF) is a noveladipokine that links obesity and inflammation,” Exp. Mol. Med.41:208-216; Alexaki, V. I., et al., 2009, “Adipocytes as immune cells:differential expression of TWEAK, BAFF, and APRIL and their receptors(Fn14, BAFF-R, TACI, and BCMA) at different stages of normal andpathological adipose tissue development,” J. Immunol. 183:5948-5956;Bossen, C., et al., 2008, “TACI, unlike BAFF-R, is solely activated byoligomeric BAFF and APRIL to support survival of activated B cells andplasmablasts,” Blood 111(3):1004-1012; Huard, B., et al., 2004, “BAFFproduction by antigen-presenting cells provides T cell co-stimulation,”Int. Immunol. 16:467-475, each of which is hereby incorporated byreference herein in its entirety.

BAFF, Receptors, and Applications Thereof

Any methods or compositions that can modulate the level and/or activityof BAFF can be used to modulate energy metabolism, in particularthermogenesis by brown fat. More precisely, any methods or compositionsthat can modulate the level and/or activity of BAFF can be used toenhance thermogenesis by brown fat, thereby achieving weight control.Any such methods and/or compositions can be used to treat and/or preventobesity.

Given BAFF and related signaling pathways and their importance inmetabolism regulation and thermogenesis, the methods and/or compositionsdescribed herein are applied directly to enhance the level and/oractivity of BAFF. For example, BAFF can be overexpressed in the targetedanimal through genetic modification (Examples 3 and 4) and can be usedto treat and prevent obesity.

BAFF is believed to bind its receptors as oligomers, for example, asheterotrimers, homotrimers, or 60-mers. BAFF of any form can be used inthe present invention.

Recombinant human BAFF is commercially available, for example, from R&DSystems, Inc. (Minneapolis, Minn.). In some embodiments, recombinantBAFF is treated with an appropriate buffer before intraperitonealdelivery. For example, mildly acidic conditions favor formation of thetrimer (e.g. around pH 6.0), while nearly neutral pH conditions (e.g.around pH 7.0) favor oligomerization and produce the 60-mer presumablyby duplicating trimers. It possible to use a delivery buffer with a pHvalue anywhere between 6.0 and 7.4 in order to achieve the formation ofthe desired trimer and 60-mer composition. In some embodiments,recombinant BAFF is genetically modified in order to preferentiallyselect for the trimer or 60-mer. Methods for making BAFF trimers andBAFF 60-mers can be found, for example, in United States PatentPublication No. 20090215071 to Cachero et al.; U.S. Pat. No. 7,553,930,each of which is hereby incorporated by reference herein in itsentirety.

In some embodiments, variants of BAFF are used in treating andpreventing obesity. For example, a shortened version of recombinanthuman BAFF of only 153 amino acids is also available as a lyophilizedprotein from BioVision (Mountain View, Calif.). Additional BAFF variantsthat can be used to treat obesity also include those disclosed in U.S.Pat. No. 7,553,930 to Desjarlais et al., United States PatentPublication No. 20060136136 by Karpusas, each which is herebyincorporated by reference herein in its entirety.

In some embodiments, antibodies to membrane-bound BAFF are used becausethey can drive signaling. In some embodiments, other agonists of BAFFare used to enhance signaling. Exemplary BAFF agonists include but arenot limited to: (a) a soluble BAFF polypeptide, (b) an agonist anti-BAFFantibody or antigen-binding fragment thereof, and (c) an agonistanti-BR3 antibody or antigen-binding fragment thereof. Examples ofsoluble BAFF peptides include but are not limited to BAFF amino acids145-284, amino acids 169-308, and amino acids 11-150.

In some embodiments, analogues of soluble BAFF peptides are used intreating and preventing obesity. For example, a peptide analoguescomprises a sequence that is at least 50% or more, 60% or more, 70% ormore, 80% or more, 90% or more, or 95% or more of any of the solubleBAFF peptide sequences. These analogues can bear 50% or more, 60% ormore, 70% or more, 80% or more, 90% or more, or 95% or more sequencehomology to those soluble fragments of BAFF. In turn, the solublefragments of BAFF can be 5% or more, 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,90% or more, or 95% or more of the full length of the BAFF protein.FIGS. 7A-C depict exemplary amino acid sequences of BAFF (SEQ ID NOs1-3).

Additional examples of BAFF agonists can be found, for example, inUnited States Patent Publication No. 20080254030 by Mackay et al., whichis hereby incorporated by reference herein in its entirety.

In some embodiments, overexpression of BAFF is achieved by geneticmanipulation, as described in Examples 3 and 4. In some embodiments,transcription regulators are used. For example, BAFF transcriptexpression increases by a toll-like receptor 4 (TLR4) agonist, alipopolysaccharide (LPS), reactive oxygen species (ROS) production,NF-κB activation, p65, and co-activator, p300.

In some embodiments, molecules that affect the activities of these BAFFtranscription regulators can be used to modulate the expression level ofBAFF. See, for example, Moon, E. and Park, H., 2007, “B cell activatingfactor (BAFF) gene promoter activity depends upon co-activator, p300,”Immunobiology 212(8):637-645, which is hereby incorporated by referenceherein in its entirety.

In some embodiments, recombinant BAFF can be introduced into a targetanimal by either gene delivery or protein delivery. For example, avector encoding BAFF can be introduced into a target animal followed byinduction of its expression with specification compounds.

It will be understood by one of skill in the art that overexpressingBAFF may also lead to adverse side effects beside the desired protectionagainst weight gain. For example, BAFF has been implicated in autoimmunediseases. However, the levels of BAFF required for triggering autoimmunedisease are much higher than those required for modulating a thermogenicresponse and/or modulating weight.

In some embodiments, the methods and/or compositions are appliedindirectly to BAFF, though an upstream or downstream effector of BAFF.For example, an inhibitor of BAFF can be removed permanently (e.g. viagenetic knockout), temporarily curtailed (e.g. by using RNAi agentstargeting the BAFF inhibitor to cause temporary reduced expression, orby using small molecule compounds that bind to the BAFF inhibitor toachieve the same effects).

In some embodiments, modulation of BAFF activity is performed bymodulating any of the factors that interact with BAFF, including but arenot limited to BAFF-R, TALI, BCMA, and APRIL, a relative of BAFF whichcompetes with BAFF in binding to TACT and BCMA. BAFF's functional rolein energy metabolism and weight control is believed to have beenachieved through one or more pathways involving these receptors.

BAFF-R, or tumor necrosis factor receptor superfamily member 13C(TNFRSF13), is a protein that in humans is encoded by the TNFRSF13Cgene, which interacts with BAFF. BAFF enhances B-cell survival in vitroand is a regulator of the peripheral B-cell population. Overexpressionof BAFF in mice results in mature B-cell hyperplasia and symptoms ofsystemic lupus erythematosus (SLE). Also, some SLE patients haveincreased levels of BAFF in serum. Therefore, it has been proposed thatabnormally high levels of BAFF may contribute to the pathogenesis ofautoimmune diseases by enhancing the survival of autoreactive B-cells.The protein encoded by this gene is a receptor for BAFF and is a typeIII transmembrane protein containing a single extracellularphenylalanine-rich domain. It is thought that this receptor is theprincipal receptor required for BAFF-mediated mature B-cell survival.More details on BAFF-R can be found in Thompson, J. et al., 2001,“BAFF-R, a newly identified TNF receptor that specifically interactswith BAFF,” Science 293(5537):2108-2111; U.S. Pat. No. 7,635,677 toAmbrose et al., each of which is hereby incorporated by reference hereinin its entirety.

TACI, also known as tumor necrosis factor receptor superfamily member13B (TNFRSF13B), is a transmembrane receptor protein found predominantlyon the surface of B cells, which are an important part of the immunesystem. TACI is a lymphocyte-specific member of the TNF receptorsuperfamily. It was originally discovered due to its ability to interactwith calcium-modulator and cyclophilin ligand (CAML). TACI was laterfound to play a crucial role in humoral immunity by interacting with twomembers of the TNF family, namely BAFF and APRIL. These proteins signalthrough TACI, inducing activation of several transcription factors,including NFAT, AP-1, and NF-κB, which then modulate cellularactivities. Defects in the function of TACI can lead to immune systemdiseases.

In some embodiments, a pharmaceutical composition comprising oligomericforms of BAFF and APRIL can be used to activate TACI-based signaling.This is due to the activation of TACI-based signaling by oligomericforms of BAFF and APRIL.

BCMA, or tumor necrosis factor receptor superfamily member 17, is aprotein that in humans is encoded by the TNFRSF17 gene. BCMA is a memberof the TNF-receptor superfamily. This receptor is preferentiallyexpressed in mature B lymphocytes, and may be important for B-celldevelopment and autoimmune response. This receptor has been shown tospecifically bind to the TNF (ligand) superfamily, member 13b(TNFSF13B/TALL-1/BAFF), and to lead to NF-κB and MAPK8/JNK activation.This receptor also binds to various TRAF family members and thus cantransduce signals for cell survival and proliferation.

APRIL is another TNF-like cytokine that stimulates tumor cell growth.Within the TNF ligand superfamily, APRIL is most similar to B-cellactivation factor (BAFF), with which it shares 30% sequence identity.APRIL binds the B-cell maturation antigen (BCMA) and TACI receptors withhigh affinity; both of these receptors have also been shown to bindBAFF, although BCMA has significantly higher affinity for APRIL thanBAFF.

In some embodiments, cell-signaling pathways parallel to the BAFFpathway, such as the APRIL pathway, are targeted for treating andpreventing obesity. APRIL signaling can be synergistic to the effect ofBAFF signaling. Similarly, any methods and/or composition designsapplicable to BAFF can be used in formulating any composition based onAPRIL. For example, recombinant APRIL and variants thereof can bedelivered directly to a subject. Additionally soluble fragments of APRILand analogues thereof can also be used in the present invention. Theseanalogous can bear 50% or more, 60% or more, 70% or more, 80% or more,90% or more, or 95% or more sequence homology to those soluble fragmentsof APRIL. In turn, the soluble fragments of APRIL can be 5% or more, 10%or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, 90% or more, or 95% or more of the fulllength of the APRIL protein. FIGS. 7D-E depict exemplary amino acidsequences of APRIL (SEQ ID NOs 4 and 5).

One of skill in the art would understand that pharmaceuticalcompositions containing BAFF- or APRIL-relevant compositions can be usedin combination with each other or in sequence with each other. One ofskill in the art would understand that any methods applicable fordesigning compositions relating to BAFF are also applicable fordesigning compositions relating to APRIL.

In some embodiments, the interaction between BAFF and a receptor isenhanced by overexpression of one or more of the receptors. For example,it has been shown that IFN-γ promotes BAFF-R promoter activity andup-regulates BAFF-R mRNA expression. See Yuan, H. et al., 2010,“Characterization of the 5′-flanking region and regulation oftranscription of human BAFF-R gene,” DNA Cell Biol. 29(3):133-139, whichis hereby incorporated by reference herein in its entirety.

In some embodiments, the interaction between BAFF and a receptor isenhanced by removing an inhibitor or a molecule that interferes with theinteraction. For example, BAFF-R appears to be selective for BAFF, butAPRIL competes with BAFF in binding to TACI and BCMA. In someembodiments, the interaction between BAFF and TACI or BCMA will beenhanced by selectively removing the interference from APRIL. See,Schneider, P. and Tschopp, J., 2003, “BAFF and the regulation of B cellsurvival,” Immunology Letters 88(1): 57-62, which is hereby incorporatedby reference herein in its entirety. In some embodiments, antibodiestargeting APRIL can be used to enhance the interaction between BAFF anda receptor.

In some embodiments, the antibody targeting a receptor of BAFF or theBAFF-receptor complex is a monoclonal antibody or a polytonal antibody.In some embodiments, the interaction between BAFF and a receptor isenhanced by prolonging the interaction. For example, a molecule can beadded to prevent the dissociation of the BAFF-receptor complex in orderto substantiate the downstream effects, such as protection againstweight gain and/or glucose dysmetabolism.

In alternative embodiments, analogues of BAFF can be added which wouldbind to one or more the BAFF receptors to initiate the desireddownstream events. The analogue of BAFF can be a peptide based on one ofmore functional sequences of BAFF. In some embodiments, peptideanalogues of BAFF are used, which comprises one or more chemicalmodifications and/or sequence modification such that the peptideanalogues are no longer identical to the natural sequence of BAFF.

In some embodiments, also provided herewith are methods and systems foridentifying factors that interact with BAFF. As mentioned above, BAFF isthe natural ligand of three TNF receptors, namely the BAFF receptor(BAFF-R, or TNFRSF13), transmembrane activator and calcium modulator andcyclophilin ligand interactor (TALI), and B-cell maturation antigen(BCMA), all of which have differing binding affinities for it. All threereceptors have recently been shown to be expressed by human adipocytes(both white and brown), as well as by mouse adipocyte cell lines.

In some embodiments, a pharmaceutical composition, such as recombinantBAFF, can be introduced directly to an animal or a human subject. Themanner of administration of a pharmaceutical composition of the presentinvention can depend upon the particular purpose for the delivery (e.g.treatment of disease or prevention of graft rejection), the overallhealth and condition of the recipient and the judgment of the physicianor technician administering the pharmaceutical composition. Apharmaceutical composition of the present invention can be administeredto an animal using a variety of methods. Such delivery methods caninclude parenteral, topical, oral, or local administration, such asintradermally or by aerosol. A pharmaceutical composition can beadministered in a variety of unit dosage forms depending upon the methodof administration. For example, unit dosage forms suitable for oraladministration to the intestinal region of an animal include powders,tablets, pills, and capsules. Preferred delivery methods for apharmaceutical composition of the present invention include intravenousadministration and local administration by, for example, injection ortopical administration. Injection can be, for example, intraperitonealinjection and/or subcutaneous injection. For particular modes ofdelivery, a pharmaceutical composition of the present invention can beformulated in an excipient of the present invention. A therapeuticreagent of the present invention can be administered to any animal,preferably to mammals, and more preferably to humans.

In some embodiments, a pharmaceutical composition of the presentinvention is administered by itself. Alternatively, a pharmaceuticalcomposition of the present invention is administered in combination withother therapeutic reagents. In some embodiments, multiple pharmaceuticalcompositions can be administered in combination with each other. Forexample, multiple BAFF peptides and/or peptide analogues can be used incombination with each other. In some embodiments, one or more peptidesand/or peptide analogues targeting APRIL can be used in combination withthose targeting BAFF.

In some embodiments, a pharmaceutical composition of the presentinvention is delivered as part of a “drug delivery system” or the like.For example, a pharmaceutical composition of the present inventionrefers to a unit dosage form of a drug or agent composition, preferablyany compositions comprising combinations of at least two or more agents,including carriers, enhancers, and other components, in which themulti-agent compound is contained in or accompanied by means formaintaining the drug composition in a drug transferring relationship orproviding any multi-agent compounds to the desired site in the body.Such means can be a patch, tablet, troche, or other device of determinedphysical form for continuous agent administration thereto for systemictransport, or such means can be formulated in free form to be applieddirectly to the patient as a cream, gel, gum, ointment, or the like.

In some embodiments, a pharmaceutical composition of the presentinvention further includes pharmaceutically acceptable adjuvants and/orcarriers. The compositions may also include other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents and otherpharmaceutical preparations known to those skilled in the art. Suchagents are known to those skilled in the art and are generally describedas being biologically inactive and can be administered to patientswithout causing deleterious interactions with the therapeutic agent.Examples of carriers or excipients for oral administration include cornstarch, lactose, magnesium stearate, microcrystalline cellulose, stearicacid, povidone, dibasic calcium phosphate, and sodium starch glycolate.Any carrier suitable for the desired administration route iscontemplated by the present invention. Additional examples of acceptableadjuvants and/or carriers include but are not limited to hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxy ethylcellulose,ethylcellulose, carboxymethyl cellulose, dextran, guar gum, polyvinylpyrrolidone, pectins, starches, gelatin, casein, acrylic acid polymers,polymers of acrylic acid esters, acrylic acid copolymers, vinylpolymers, vinyl copolymers, polymers of vinyl alcohols, alkoxy polymers,polyethylene oxide polymers, polyethers, and mixtures thereof and thelike.

In some embodiments, peptides and/or analogues of BAFF or APRIL of thepresent invention are delivered to a subject (e.g. a human in need oftreatment or at risk of developing obesity) by using one or more nucleicacids encoding the corresponding amino acid sequence.

Additional receptors and ligand interactions can also be used tomodulate thermogenesis by brown fat and can be identified by acombination of biochemical and genetic analysis. For example, DNAmicroarray technology can be used to identify proteins that areregulated by BAFF and APRIL signaling in brown fat and can also be usedto identify downstream effectors post BAFF-receptor interactions.

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that all examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrateembodiments of the invention disclosed herein. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches that have been found tofunction well in the practice of the invention, and thus can beconsidered to constitute examples of modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Functional Analysis of RP105

RP105, which has been described as a B-cell-specific molecule thatdrives proliferation when cross-linked by antibodies, was studied, withresults showing that: (a) RP105 expression mirrors that of TLRs onmyeloid and other cells; and (b) RP105 inhibits TLR4 signaling inprimary myeloid cells and cell lines and regulates in vivo responses toLPS. Preliminary data indicate that RP105 also regulates energymetabolism in response to caloric excess. The present experiments alsoshowed that RP105-deficient mice are protected from obesity and glucosedysmetabolism.

Six to eight week-old male C57BL/6 RP105−/− mice and wild type (WT)controls were placed on a high fat diet (HFD) or a regular chow diet(RD). As shown in FIG. 1A, RP105-deficient mice gain significantly lessweight than WT controls when subjected to HFD stress. RP105−/− mice eatthe same amount as WT controls on a HFD and have identical foodabsorption data (as quantified by bomb calorimetry), which is indicativeof an effect on the regulation of energy metabolism.

Adaptive thermogenesis by brown fat, which occurs in response to coldstress as well as dietary excess, is dependent on the expression ofUCP1. The data demonstrate that: (a) brown fat from RP105−/− mice on aHFD express significantly more UCP1 compared to WT controls (data notshown); and (b) the obesity phenotype of RP105−/− mice on a HFD is evenmore robust under thermoneutral conditions. The latter finding suggeststhat the relevant variable is dietary excess, rather than the coldstress associated with standard housing. As might be expected, inparallel with protection from obesity, RP105−/− mice are protected fromthe metabolic and end-organ sequelae of obesity, including glucosedysmetabolism (as shown in FIG. 1B), dyslipidemia, and hepaticsteatosis. These effects upstream of the development of obesity arecompatible with the described role of TLRs in driving these downstreamsequelae.

FIG. 1B shows fasting glucose levels, quantified after the mice had beenon a designated diet for 10 weeks. After an 8 hour fast, blood glucoselevels were quantified by glucometer. RD results are depicted by whitebars; HFD results are depicted by black bars. Data represent means±SE ina single experiment, representative of 10 individual experiments;n=10-14 mice/group. For FIG. 1A, AUC ANOVA P<0.01, Tukey's correction***P<0.001; for FIG. 1B, ANOVA P<0.0001, Tukey's correction *P<0.01.

Example 2 Effect of RP105 on LPS-Driven B-Cell Proliferation

RP105-deficient mice exhibit blunted LPS-driven B cell-proliferation,something that had been interpreted to indicate that RP105 facilitatesTLR4 signaling in B-cells. Given the fact that RP105 inhibits TLR4signaling in other cells, the role of RP105 in B-cell function wasre-examined. It was found that: (a) modulation of TLR4-driven B-cellproliferative responses by RP105 is not a function of B-cell-intrinsicexpression of RP105; and (b) a mechanistic role for dysregulated BAFFexpression was demonstrated. As RP105−/− mice exhibit significantlyincreased basal and TLR-driven BAFF expression (as shown in FIG. 2A),transgenic overexpression of BAFF recapitulates the impaired TLR4-drivenproliferation by RP105−/− B-cells, and partial neutralization of BAFFrescues the aberrant B-cell proliferative responses of RP105−/− mice.These data also elucidate into the role of RP105 in regulating energymetabolism.

BAFF was discovered for its role in B-cell maturation and survival. BAFFoverexpression augments B-cell numbers and can lead to the developmentof B-cell-dependent autoimmune disease. In contrast, genetic deletion orantibody-mediated inhibition of BAFF results in contraction of theB-cell compartment. BAFF is produced by diverse myeloid, lymphoid andstromal cell types, including adipocytes.

FIG. 2 illustrates the comparison among serum BAFF levels in RP105−/−,μMT, and BAFF-Tg mice. Among other stimuli, TLR signaling induced BAFFexpression (as shown in FIG. 2A). For the kinetic analysis, mice weremock-challenged or challenged with TLR4-specific E. coli LPS (40 ng,i.p.), and serum BAFF levels were determined kinetically.

BAFF binds to 3 receptors, namely the BAFF receptor (BAFF-R),transmembrane activator and calcium modulator and cyclophilin ligandinteractor (TALI), and B-cell maturation antigen (BCMA). All 3 receptorshave recently been shown to be expressed by human adipocytes (both whiteand brown), as well as by mouse adipocyte cell lines.

Example 3 Effect of BAFF Concentration

BAFF overexpression was shown to be associated with protection fromweight gain and glucose dysmetabolism. In the experiments shown in FIG.3, six to eight week-old male μMT, BAFF-transgenic, and WT mice (all ona C57BL/6 background) were placed on a HFD or RD.

B-cell-deficient (μMT) mice exhibited robust secondary increases incirculating BAFF concentrations (in response to the lack of B-cells),and BAFF-transgenic (BAFF-Tg) mice had extremely robust serum BAFFlevels (FIG. 2B). The responses of these mice to HFD stress weredefined.

Similar to RP105−/− mice (as shown in FIG. 1), μMT and BAFF-Tg mice weresignificantly protected from developing obesity on a HFD (as shown inFIGS. 3A and 3C). In these experiments, (a) mouse weight correlateddirectly with adiposity (data not shown); and (b) protection fromobesity was associated with protection from glucose dysmetabolism (asshown in FIGS. 3B and 3D). FIGS. 3B and 3D depict fasting glucoselevels, quantified after the mice had been on a designated diet for 9weeks.

Example 4 Response to HFD Stress

In comparison with WT mice, genetically modified mice with higher levelsof BAFF showed more resistance to weight gain when they were subjectedto the stress of a HFD. The data suggest an approximate log-linear doseresponse in terms of the effects of circulating BAFF levels on weightgain. For example, FIG. 2B shows that μMT mice exhibited serum BAFFlevels 1.5 log higher than that of the RP105−/− mice, and BAFF-Tg miceexhibited a further 1.5 log elevation in serum BAFF levels. FIG. 4further demonstrates that the difference in levels of BAFF correlatewith differences in weight gain by the different types of mice. Thesedata indicate that the modest elevations of BAFF observed in RP105−/−mice were sufficient for significant effects on obesity.

Example 5 Brown Adipocyte Respiration

Given the data on BAFF expression and the response to HFD stress, thedata indicating increased UCP1 expression by brown fat from RP105−/−mice on a HFD, and recent reports of BAFF receptor expression by brownfat, the potential direct effects of BAFF on brown adipocyte respirationwere studied. As shown in FIG. 5, in vitro treatment of an adipocytecell line with recombinant BAFF resulted in rapid up-regulation inrespiration by brown adipocytes.

Example 6 Analysis of Brown Fat Mitochondria

Brown fat mitochondria from RP105-deficient mice exhibit increaseduncoupled respiration (as shown in FIG. 6). WT and RP105−/− C57BL/6 malemice were fed a HFD for 24 weeks. Uncoupled respiration(pyruvate/malate-induced, GDP inhibitable respiratory activity) wasquantified in mitochondria isolated from brown adipose tissue using anXF24 extracellular flux analyzer (Seahorse Bioscience).

Example 7 Increased Norepinephrine-Induced Thermogenesis inRP105-Deficient Mice

Adaptive thermogenesis was compared in RP105−/− and WT mice on a HFD.RP105−/− and WT mice, both on a C57BL/6 background, were placed on a HFD(60% kcal from fat, 20% kcal from carbohydrate, 20% kcal from protein,Research Diets #D124920 for 14 days. Mice were anaesthetized byintraperitoneal injection of ketamine/xylazine, and the basal rate ofoxygen consumption (at 33° C.) was quantified kinetically, at 3 minuteintervals for 24 minute, by indirect calorimetry. Oxygen consumptioncontinued to be quantified kinetically after subsequent subcutaneouschallenge, depicted on FIG. 8 with an arrow, with 1 mg/kg norepinephrinebitartrate. WT results are represented by open symbols; RP105−/− resultsare represented by closed symbols. Data represent means+/−SE of an n=4-5mice/experimental group; t test; *P<0.05.

Example 8 Up-Regulated Norepinephrine-Induced Thermogenesis as a Resultof BAFF Treatment

WT C57BL/6 mice were placed on either a HFD (60% kcal from fat, 20% kcalfrom carbohydrate, 20% kcal from protein, Research Diets #D124920 or aRD (chow; 13.5% kcal from fat, 59% kcal from carbohydrate, 27.5% kcalfrom protein, Lab Diet #5010) for 14 days. Mice were subsequently given1 mg/kg recombinant BAFF or vehicle control (saline) by intraperitonealinjection 18 hours and 2 hours prior to quantification of oxygenconsumption. Mice were then anaesthetized by intraperitoneal injectionof ketamine/xylazine, and the basal rate of oxygen consumption (at 330C) was quantified kinetically, at 5 minute intervals for 25 minutes, byindirect calorimetry. Oxygen consumption continued to be quantifiedkinetically after subsequent subcutaneous challenge, depicted on FIG. 9with an arrow, with 1 mg/kg norepinephrine bitartrate. RD+saline resultsare represented by open squares; RD+BAFF results are represented byclosed squares; HFD+saline results are represented by open circles;HFD+BAFF results are represented by closed circles. Data representsingle mice/experimental group.

Example 9 Improved Maintenance of Body Core Temperature byRP105-Deficient Mice During Cold Exposure

The ability of RP105-deficient mice to maintain body core temperatureduring cold exposure was studied. Six to eight week-old male C57BL/6RP105^(−/−) mice and WT controls were placed on a HFD for 16 weeks.Animals were subsequently placed at 4° C., and body core temperature wasquantified. The mice were then subjected to 0-6 hours of cold exposure,under lighted conditions; 6-12 hours cold exposure was conducted underdark conditions. FIG. 10 illustrates that RP105-deficient mice maintainbody core temperature better during cold exposure. WT HFD results arerepresented by white squares; RP105^(−/−) HFD results are represented byblack squares. All data represent means±SE in a single experiment,representative of 2 individual experiments; n=5-6 mice/group; *P<0.05(Student's t test).

Example 10 Increased UCP-1 Expression in Brown Adipose Tissue ofRP105-Deficient Mice

The expression of UCP-1 in the brown adipose tissue of RP105-deficientmice was then studied. Six to eight week-old male C57BL/6 RP105^(−/−)mice and wild type (WT) controls were placed on a regular diet (RD) or ahigh fat diet (HFD) for 20 weeks, and UCP-1 expression was quantified byqRT-PCR. FIG. 11 illustrates that RP105-deficient mice have increaseduncoupling protein 1 (UCP-1) expression in brown adipose tissue. WTresults are represented by white bars; RP105^(−/−) results arerepresented by black bars. All data represent means±SE in a singleexperiment, representative of 2 individual experiments; n=6 mice/group;*P<0.05 (Student's t test).

The various methods and techniques described above provide a number ofways to carry out the invention. Those skilled in the art will recognizethat the methods can be performed in a manner that achieves or optimizesone advantage or group of advantages as taught herein withoutnecessarily achieving other objectives or advantages as may be taught orsuggested herein. It is to be understood that some embodimentsspecifically include one, another, or several features, while othersspecifically exclude one, another, or several features, while stillothers specifically mitigate a present disadvantageous feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

In some embodiments, the numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the scope of someembodiments of the invention are approximations, the numerical valuesset forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

1. A method of treating or preventing diet-induced obesity and/or one ormore sequelae thereof in a subject, comprising: administering to asubject in need of treatment or at risk for developing obesity and/orone or more sequelae thereof, a therapeutically effective amount of apharmaceutical composition to enhance thermogenesis associated withbrown fat or brown fat cells, wherein said pharmaceutical composition isselected from the group consisting of a recombinant B-cell activatingfactor (BAFF) or a variant thereof, a BAFF-based peptide or peptideanalogue, a BAFF transcription regulator, a nucleic acid encoding atleast a partial sequence of BAFF, a first factor upstream or downstreamfrom the interaction between BAFF and a first receptor, aproliferation-inducing ligand (APRIL) that is recombinant or a variantthereof, an APRIL-based peptide or peptide analogue, an APRILtranscription regulator, a nucleic acid encoding at least a partialsequence of APRIL, a second factor upstream or downstream from theinteraction between an APRIL and a second receptor, a regulator of aBAFF-first receptor interaction or an APRIL-second receptor interaction,and a combination thereof.
 2. The method of claim 1, whereinthermogenesis associated with brown fat comprises thermogenesis by brownfat or brown fat cells, adaptive thermogenesis by brown fat or brown fatcells, thermogenic capacity of brown fat or brown fat cells, or acombination thereof.
 3. The method of claim 1, wherein thermogenesisassociated with brown fat or brown fat cells is enhanced by increasingthe level or activity of BAFF.
 4. The method of claim 3, whereinthermogenesis associated with brown fat or brown fat cells is furtherenhanced by increasing the level or activity of APRIL.
 5. The method ofclaim 1, wherein said first receptor of BAFF is a BAFF-receptor(BAFF-R), cytophilin ligand interactor (TACI) or B-cell maturationantigen (BCMA).
 6. The method of claim 1, wherein said pharmaceuticalcomposition is a regulator of a BAFF-first receptor interaction selectedfrom the group consisting of a BAFF-based peptide or an analoguethereof, a BAFF receptor analogue, a co-activator of a BAFF-firstreceptor interaction, and a combination thereof.
 7. The method of claim1, wherein said pharmaceutical composition is a recombinant BAFF or avariant thereof.
 8. The method of claim 1, wherein said first factorupstream or downstream from the interaction between BAFF and said firstreceptor is radioprotective protein 105 kDa (RP105).
 9. The method ofclaim 1, wherein said pharmaceutical composition is administered to asubject via parenteral, topical, oral, or local administration,injection, or a combination thereof.
 10. The method of claim 9, whereinsaid subject is a human in need of treatment or at risk for developingobesity and/or one or more sequelae thereof.
 11. The method of claim 1,further comprising: identifying said subject in need of treatment or atrisk for developing obesity and/or one or more sequelae thereof.
 12. Themethod of claim 1, wherein said BAFF transcription regulator comprisestoll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS), reactiveoxygen species (ROS) production and NF-κB activation, p65, co-activatorp300, or a combination thereof.
 13. The method of claim 1, wherein saidsecond receptor of APRIL is TACI or BCMA.
 14. The method of claim 1,wherein thermogenesis associated with brown fat or brown fat cells isenhanced by increasing the level or activity of APRIL.
 15. The method ofclaim 1, wherein thermogenesis associated with brown fat or brown fatcells is enhanced by increasing the level or activity of APRIL, BAFF, orsome combination thereof.
 16. The method of claim 1, wherein saidpharmaceutical composition is a regulator of APRIL-second receptorinteraction selected from the group consisting of an APRIL-based peptideor an analogue thereof, an APRIL receptor analogue, and a co-activatorof an APRIL-second receptor interaction.
 17. The method of claim 1,wherein said first receptor and said second receptor are the same. 18.The method of claim 1, wherein said pharmaceutical composition is arecombinant APRIL, a fragment of APRIL, an APRIL variant, or acombination thereof.